High affinity t cell receptor and use thereof

ABSTRACT

The present invention is directed to a high affinity T cell receptor (TCR) against a tumor-associated antigen, an isolated nucleic acid molecule encoding same, a T cell expressing said TCR, and a pharmaceutical composition for use in the treatment of diseases involving malignant cells expressing said tumor-associated antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/822,970, filed Nov. 27, 2017 (now U.S. Pat. No. 10,626,159), which isa divisional of U.S. patent application Ser. No. 14/224,525, filed Mar.25, 2014 (now U.S. Pat. No. 9,862,755), which is a divisional of U.S.patent application Ser. No. 13/130,665; filed May 23, 2011 (now U.S.Pat. No. 8,697,854); which itself is a National Stage Entry of PCTInternational Patent Application Serial No. PCT/EP2009/065705, filedNov. 24, 2009; which itself claims the benefit of European PatentApplication Serial No. EP 08020396.1, filed Nov. 24, 2008. Thedisclosure of each of these applications is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to a high affinity T cell receptor(TCR) against a tumor-associated antigen, an isolated nucleic acidmolecule encoding same, a T cell expressing said TCR, and apharmaceutical composition for use in the treatment of diseasesinvolving malignant cells expressing said tumor-associated antigen.

BACKGROUND OF THE INVENTION

TCR's are members of the immunoglobulin superfamily and usually consistof two subunits, namely the α- and β-subunits. These possess oneN-terminal immunoglobulin (Ig)-variable (V) domain, one Ig-constant (C)domain, a transmembrane/cell membrane-spanning region, and a shortcytoplasmic tail at the C-terminal end. The variable domains of both theTCR α-chain and β-chain have three hypervariable or complementaritydetermining regions (CDRs), whereas the variable region of the β-chainhas an additional area of hypervariability (HV4) that does not normallycontact antigen and therefore is not considered a CDR.

CDR3 is the main CDR responsible for recognizing processed antigen,although CDR1 of the alpha chain has also been shown to interact withthe N-terminal part of the antigenic peptide, whereas CDR1 of theβ-chain interacts with the C-terminal part of the peptide. CDR2 isthought to recognize the MHC. CDR4 of the β-chain is not thought toparticipate in antigen recognition, but has been shown to interact withsuperantigens. The constant domain of the TCR domain consists of shortconnecting sequences in which a cysteine residue forms disulfide bonds,which forms a link between the two chains.

The affinity of TCR's for a specific antigen makes them valuable forseveral therapeutic approaches. For example, cancer patients, such asmelanoma patients, can be effectively treated by using adoptiveimmunotherapy.

The adoptive transfer of lymphocytes in the setting of allogeneic stemcell transplantation (SCT) has demonstrated the power of the immunesystem for eradicating hematological malignancies (Kolb et al. 1995). Itappears that SCT can also function to eliminate solid tumors, such asrenal cell carcinomas (RCC) in some cases (reviewed in Kolb et al. 2004and Dudley and Rosenberg, 2003). In SCT recipients, the elimination ofmalignant cells may only occur after several months up to a year, due tothe fact that specific T cells must be activated in vivo and must thenexpand to adequate numbers following the development of the newhematopoietic system in the transplant recipient. Alternatively, after aperiod of time (approximately 60 days) during which tolerance isestablished in the SCT recipient, a transfer of unprimed, unseparatedlymphocytes can be made to speed up the generation of immune responsesdirected against tumor cells. Here again, the specific lymphocytescapable of attacking tumor cells must be activated and expanded from thelow frequency precursor lymphocytes that are present among theunselected population of lymphocytes that are transferred. Donorlymphocyte infusions (DLI) of unselected lymphocyte populations afterSCT work well for the elimination of chronic myelogenous leukemia (CML),which grows slowly, but are less effective in the eradication of acuteleukemia, partly due to the fact that the growth of the malignant cellsoutpaces the expansion capacity of the immune cells. This same expansiondifferential in which immune cells expand more slowly than tumor cells,also impacts on the poor immune elimination of rapidly progressing solidtumors. A second handicap in the use of unselected mixed lymphocytepopulations in DLI is that T cells may also be transferred that have thecapacity to attack normal cells and tissues of the recipient, leading tograft-versus-host-disease (GVHD), a disease with high morbidity andmortality.

Recent studies have demonstrated that the adoptive transfer of selectedT cells with defined peptide specificities can lead to major reductionsin tumor burden in an autologous setting, particularly if patients havebeen pretreated with non-myeloablative regimens (Dudley et al. 2002,2003). This eliminates the need to perform SCT in the tumor patient, andthereby also bypasses the problem of GVHD.

In order to extend the capacity to use adoptive cell therapy (ACT) totreat patients with more rapidly growing tumors, it is a goal totransfer enriched, peptide-specific effector T cells (both CD4 T helpercells and cytotoxic T lymphocytes) that have been selected for theirligand specificities to effectively attack tumor cells while avoidingserious attack of normal tissues. These cells are to be rapidly expandedto large numbers ex vivo and then used for ACT. Alternatively, the Tcell receptors (TCR) of such ligand-specific T cells can be cloned andexpressed as TCR-transgenes in activated lymphocytes, using eitherrecipient peripheral blood lymphocytes or activated T cell clones withdefined specificities that grow well and do not have the capacity toattack normal host tissues.

As examples, an expanded allospecific T cell clone that is specific foran MHC molecule not expressed by the recipient or an expanded T cellclone specific for a virus, such as cytomegalovirus or Epstein-Barrvirus, could be used as recipient cells for the transgenic TCR. Theavailability of a panel of transgenic TCR vectors, recognizing differentMHC-peptide ligands could be used to develop large numbers ofpre-activated T cells of both the CD4 and CD8 subtypes, thereby allowinglarge numbers of effector lymphocytes to be rapidly prepared andtransferred to patients whose tumors express the corresponding TCRligands. This would save time in achieving the numbers of specific Tcells required to control tumor growth, possibly leading to moreeffective tumor eradication of rapidly progressing tumors.

Because the determinants that specific T cells recognize on leukemia andlymphomas, as well as solid tumor cells, often represent self-peptidesderived from over-expressed proteins that are presented by self-MHCmolecules, the affinity of their T cell receptors (TCR) is low, since Tcells bearing high affinity receptors have been eliminated through theprocess of negative selection which is applied to lymphocytes duringtheir development in the thymus to prevent autoimmunity. More effectivetumor cell recognition occurs if the T cells are generated fromlymphocyte populations that have not been negatively selected againstself-MHC-molecules during their development in the thymus.

WO 2006/031221 pertains to T cell receptors against tumor-associatedantigens, nucleic acids encoding the same, vectors and cells comprisingthe nucleic acids encoding the T cell receptors, and methods of usethereof. Among others, it is disclosed that the TCR subunits have theability to form TCR that confer specificity to T cells for tumor cellspresenting MART-I, NY-ESO-I, and melanoma-related gp100.

In the prior art, several scientific and patent documents are existing,which describe TCR, which are able to recognise and bind tyrosinase.Visseren et al. (Int. J. Cancer (1997) 72, 1122-1128) describe theaffinity and specificity of several tyrosinase-specific TCR and suggestto use these TCR as a specific treatment of melanoma patients.

Roszkowski et al. (J. Immunol. (2003) 170, 2582-2589 and Cancer Res.(2005) 65, 1570-1576) the like are characterising tyrosinase-specificTCR.

U.S. Pat. No. 5,906,936 is directed to cytotoxic T-cells which killnon-MHC-restricted target cells and not to cells, which comprisespecific TCR sequences.

WO97/32603 is directed to a method for producing non-human TCR and TCRspecific for human HLA-restricted tumor antigens. Furthermore, theTCR-nucleic acids and recombinant T-cells are described as well as theadministration of TCR recombinant T-cells for the treatment of severaldiseases.

WO2007/065957 describes an effector T-cell transfected with an antigenspecific TCR coding RNA wherein the transfected T-cell recognizes theantigen in a complex with the MHC-molecule and binds the same. As apotential tumor antigen, MART-1 (Melan-A), tyrosinase and survivin arenamed.

WO2008/039818 discloses MART-1 and tyrosinase-specific TCR sequences anddescribes the enhancement of antigen recognition by substitution in theCDR2 region.

The above prior art TCR sequences are all derived from autologous orxenogeneic, but not allogeneic, sources.

For example, TCR sequences are from peripheral blood or from tumorinfiltrating lymphocytes of HLA-A2 positive melanoma patients. Thismeans that all these TCR are HLA-A2 self-restricted TCRs, or, areHLA-DP4 restricted, NY-ES 0-1 specific, both derived from autologoussources. As an alternative, as disclosed in WO97/32603, the TCR isderived from an HLA-A2 transgenic mouse, so in this case the sequence isxenogeneic.

However, the available prior art documents do not show TCR sequences,which are allo-restricted and tyrosinase-specific.

Thus, there is still an important need to find means to generate T cellsthat bear TCR with high functional avidity that have the capacity torecognize specific ligands on tumor cells. Although adoptive transfer ofT cells expressing transgenic T cell receptors (TCR) with anti-tumorfunction is a hopeful new therapy for patients with advanced tumors,there is a critical bottleneck in identifying high-avidity T cells withTCR specificities needed to treat different malignancies.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide TCR orfunctional parts thereof, such as CDR3 regions, which show high affinityagainst tumor-associated antigens, in particular tyrosinase. It is afurther object of the invention to provide pharmaceutical compositionsfor use in adoptive cell therapy which allow an effective treatment ofdiseases involving malignant cells expressing tyrosinase, preferablymelanomas, gliomas, glioblastomas, and/or rare tumors of ectodermalorigin.

These objects are solved by the subject-matter of the independentclaims. Preferred embodiments are indicated in the dependent claims.

TCR specific for the melanoma-associated antigen, tyrosinase, could beisolated by the inventors and it could be shown that TCR derived fromthe allo-restricted clone were superior in recognition of specificpeptide and tumor cells after expression as transgenes in recipientlymphocytes. Therefore, TCR's and functional parts thereof, such as CDR3regions could be identified, which find application in adoptive celltherapy for the treatment of several malignancies.

A number of T cell clones with specificity for various tumor-associatedantigens have been reported over the years (see above). Most of theseTCR are restricted by self-MHC molecules. The TCR sequences disclosedherein, however, are allo-restricted and show high-avidity inrecognition of their specific ligands. The TCR of the present inventionare not self-MHC-restricted and therefore have higher structuralaffinity for interactions with MHC-peptide ligands that target tumorcells via common over-expressed self proteins. As it will be outlined inthe EXAMPLES, the TCR of the present invention were derived from a Tcell clone generated by priming CD8* T cells with autologous dendriticcells from an HLA-A2 negative donor co-expressing allogeneic HLA-*A0201molecules and an antigen. As a result, the present TCR are oftherapeutic use for the treatment of HLA-A2 positive patients.

In more detail, T cell responses against tumors are often directedagainst self-MHC molecules presenting peptides derived fromover-expressed self-proteins. In general, T cells with high avidity forself-peptide/self-MHC ligands are eliminated by negative selection toprevent autoimmunity. The TCR affinity of remaining T cells specific forself-ligands is normally low, however high-avidity T cells are needed toeffectively eradicate tumors. Because negative selection is limited toself-MHC molecules, T cells that recognize allogeneic MHC molecules havenot undergone negative selection.

However, as described in the present invention if peptides are presentedby allogeneic MHC molecules, it is feasible to obtain high-avidity Tcells specific for common tumor-associated ligands derived fromover-expressed self-proteins.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the present invention provides a nucleicacid molecule coding for the V(D)J regions of a TCR that recognizes atumor antigen and comprising the nucleic acid sequence of SEQ ID NO: 1coding for the α-chain and/or comprising the nucleic acid sequence ofSEQ ID NO: 2 coding for the β-chain of said TCR.

Therefore, a TCR of the present invention and a nucleic acid sequenceencoding the same may comprise only one of the α-chain or β-chainsequences as defined herein (in combination with a further α-chain orβ-chain, respectively) or may comprise both chains.

The term “nucleic acid” as used herein with reference to nucleic acidsrefers to a naturally-occurring nucleic acid that is not immediatelycontiguous with both of the sequences with which it is immediatelycontiguous (one on the 5′end and one on the 3′end) in thenaturally-occurring genome of the cell from which it is derived. Forexample, a nucleic acid can be, without limitation, a recombinant DNAmolecule of any length, provided one of the nucleic acid sequencesnormally found immediately flanking that recombinant DNA molecule in anaturally-occurring genome is removed or absent. Thus, a nucleic acidincludes, without limitation, a recombinant DNA that exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as recombinant DNA that is incorporated into a vector,an autonomously replicating plasmid, a virus (e.g., a retrovirus, oradenovirus). In addition, an isolated nucleic acid can include arecombinant DNA molecule that is part of a hybrid or fusion nucleic acidsequence.

Furthermore, the term “nucleic acid” as used herein also includesartificially produced DNA or RNA sequences, such as those sequencesgenerated by DNA synthesis based on in silico information.

The nucleic acids of the invention can comprise natural nucleotides,modified nucleotides, analogs of nucleotides, or mixtures of theforegoing as long as they are capable of causing the expression of apolypeptide in vitro, and preferably, in a T cell. The nucleic acids ofthe invention are preferably RNA, and more preferably DNA.

Furthermore, the present invention also comprises derivatives of theabove described nucleic acid molecules, wherein, related to the aboveSEQ ID NO: 1 and 2, the sequence has been altered by additions,deletions and/or substitutions and wherein the tumor antigen recognizingcharacteristics are maintained or improved. In other words, the tumorantigen recognizing characteristics are at least maintained.

More precisely, such a derivative is coding for the α- or β-chain,wherein the chain has been altered by one or more additions or deletionsof from 1-15 amino acids, the additions or deletions being outside theCDR3 region of each chain, and/or by conservative substitutions of from1-15 amino acids. It is noted in this connection that also the CDR3region may be altered, but to a lesser extent. The definition of thoseamendments is indicated below for the derivatives of fragments codingfor the CDR3 region.

Useful changes in the overall nucleic acid sequence in particular arerelated to codon optimization and the addition of epitope tags, whichwill be explained in detail below. Such codon optimization can includeoptimization of expression levels, optimization of avidity for targetcells, or both.

In general, it should, however, be noted that the alterations should notdiminish or alter the ability of the encoded polypeptide to form part ofa TCR that recognizes tumor associated antigens in the context of anMHC, but should facilitate destruction of a cancer cell, and preferablyfacilitate the regression of a tumor, or other cancerous state.

For example, alterations can be made which lead to conservativesubstitutions within the expressed amino acid sequence. These variationscan be made in complementarity determining and non-complementaritydetermining regions of the amino acid sequence of the TCR chain that donot affect function. However, as noted above, additions and deletionsshould not be performed in the CDR3 region (for example an addition ofepitope tags).

The concept of “conservative amino acid substitutions” is understood bythe skilled artisan, and preferably means that codons encodingpositively-charged residues (H, K, and R) are substituted with codonsencoding positively-charged residues, codons encoding negatively-chargedresidues (D and E) are substituted with codons encodingnegatively-charged residues, codons encoding neutral polar residues (C,G, N, Q, S, T, and Y) are substituted with codons encoding neutral polarresidues, and codons encoding neutral non-polar residues (A, F, I, L, M,P, V, and W) are substituted with codons encoding neutral non-polarresidues. These variations can spontaneously occur, be introduced byrandom mutagenesis, or can be introduced by directed mutagenesis. Thosechanges can be made without destroying the essential characteristics ofthese polypeptides, which are to recognize antitumor antigens in thecontext of an MHC with high avidity so as to enable the destruction ofcancer cells. The ordinarily skilled artisan can readily and routinelyscreen variant amino acids and/or the nucleic acids encoding them todetermine if these variations substantially lessen or destroy the ligandbinding capacity by methods known in the art.

In a further embodiment, the present invention provides fragments of theabove nucleic acid molecules, coding for a CDR3 region of a TCRrecognizing a tumor antigen and having the nucleic acid sequence of SEQID NO: 3 or 4 or coding for the amino acid sequences of SEQ ID NO: 5 or6. Alterations in the CDR3 region will be performed according to theconsiderations described below.

The invention further provides derivatives wherein the CDR3 region hasbeen altered by one or more additions and/or deletions of an overallnumber of from 1-5 amino acids, but not more than 1-3 contiguous aminoacids and/or conservative substitutions of from 1-6 amino acids andwherein the tumor antigen recognizing characteristics are maintained orimproved.

This means, more precisely, that additions or deletions may be performedto an extent that 1-5 amino acids are added or deleted in the CDR3region. If more then one addition or deletion is performed, the overallnumber of added or deleted amino acids may not exceed 5 amino acids.Further, one single addition or deletion at one site may only be in therange of 1-3 amino acids, i.e. 1-3 contiguous amino acids, since theligand binding capacity might be deteriorated by performing largeradditions/deletions.

A preferred derivative of the nucleic acid molecule encoding the α- orβ-chain of said TCR is one, wherein the original sequence of SEQ ID NO:1 and 2 has been altered by codon optimization. A preferred example ofsuch a derivative coding for the V(D)J regions of a TCR that recognizesa tumor antigen is the nucleic acid sequence of SEQ ID NO: 7 coding forthe α-chain and the nucleic acid sequence of SEQ ID NO: 8 coding for theβ-chain of said TCR.

Codon optimization is a generic technique to achieve optimal expressionof a foreign gene in a cell system. Selection of optimum codons dependson codon usage of the host genome and the presence of several desirableand undesirable sequence motifs. It is noted that codon optimizationwill not lead to an altered amino acid sequence and, thus, will not fallunder the definition of a conservative substitution as contained in thisapplication.

In a preferred embodiment, the tumor antigen is tyrosinase. Tyrosinaseexpressing malignancies still have a high incidence, for example, around160,000 new cases of melanoma are diagnosed worldwide each year.According to a report issued by WHO, about 48,000 melanoma relateddeaths occur worldwide per year. Thus, tyrosinase is a suitable tumorantigen which can serve as a target for tumor treatment.

In a second aspect, the present invention is directed to a TCR,preferably a soluble TCR, encoded by a nucleic acid molecule as definedabove or comprising the amino acid sequences of SEQ ID NO: 5 and/or 6.

Said TCR preferably is present in the form of a functional TCR α- and/orβ-chain fusion protein, comprising:

-   -   a) at least one epitope-tag, and    -   b) the amino acid sequence of an α and/or β chain of a TCR as        defined above or encoded by a nucleic acid molecule as outlined        above,    -   wherein said epitope-tag is selected from        -   i) an epitope-tag added to the N- and/or C-terminus of said            α- and/or β-chain, or added into the α- and/or β-chain            sequence, but outside the CDR3 region,        -   ii) an epitope-tag inserted into a constant region of said            α- and/or β-chain, and        -   iii) an epitope-tag replacing a number of amino acids in a            constant region of said α- and/or β-chain.

Epitope tags are short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been addedto a living organism or to cultured cells. Detection of the taggedmolecule can be achieved using a number of different techniques.Examples of such techniques include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“Western”), and affinity chromatography. Epitopetags add a known epitope (antibody binding site) on the subject protein,to provide binding of a known and often high-affinity antibody, andthereby allowing one to specifically identify and track the taggedprotein that has been added to a living organism or to cultured cells.

In the context of the present invention, a “functional” T-cell receptor(TCR) α- and/or β-chain fusion protein shall mean an α- and/or β-chainfusion protein that, although the chain includes the epitope-tag and/orhas a tag attached to it, maintains at least substantial fusion proteinbiological activity in the fusion. In the case of the α- and/or β-chainof a TCR, this shall mean that both chains remain able to form a T-cellreceptor (either with a non-modified α- and/or β-chain or with anotherinventive fusion protein α- and/or β-chain) which exerts its biologicalfunction, in particular binding to the specific peptide-MHC complex ofsaid TCR, and/or functional signal transduction upon peptide activation.

Preferred is a functional T-cell receptor (TCR) α- and/or β-chain fusionprotein according to the present invention, wherein said epitope-tag hasa length of between 6 to 15 amino acids, preferably 9 to 11 amino acids.

Even more preferred is a functional T-cell receptor (TCR) α- and/orβ-chain fusion protein according to the present invention, wherein saidT-cell receptor (TCR) α- and/or β-chain fusion protein comprises two ormore epitope-tags, either spaced apart or directly in tandem.Embodiments of the fusion protein can contain 2, 3, 4, 5 or even moreepitope-tags, as long as the fusion protein maintains its biologicalactivity/activities (“functional”).

Preferred is a functional T-cell receptor (TCR) α- and/or β-chain fusionprotein according to the present invention, wherein said epitope-tag isselected from, but not limited to, CD20 or Her2/neu tags, or otherconventional tags such as a myc-tag, FLAG-tag, T7-tag, HA(hemagglutinin)-tag, His-tag, S-tag, GST-tag, or GFP-tag. myc, T7, GST,GFP tags are epitopes derived from existing molecules. In contrast, FLAGis a synthetic epitope tag designed for high antigenicity (see, e.g.,U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc tag can preferably beused because high quality reagents are available to be used for itsdetection. Epitope tags can of course have one or more additionalfunctions, beyond recognition by an antibody. The sequences of thesetags are described in the literature and well known to the person ofskill in art.

In the functional T-cell receptor (TCR) α- and/or β-chain fusion proteinaccording to the present invention, said fusion protein may be forexample selected from two myc-tag sequences that are attached to theN-terminus of an α-TCR-chain and/or 10 amino acids of a protruding loopregion in the β-chain constant domain being exchanged for the sequenceof two myc-tags.

In an embodiment of the present invention, the inventors inserted anamino acid sequence that corresponds to a part of the myc protein(myc-tag) at several reasonable sites into the structure of a T cellreceptor and transduced this modified receptor into T cells (seeEXAMPLES below). By introducing a tag into the TCR structure, it ispossible to deplete the modified cells by administering the tag-specificantibody to the patient.

Those functional TCR fusion proteins may be used in a method forselecting a host cell population expressing a fusion protein selectedfrom the group consisting of a fusion protein comprising a) at least oneepitope-providing amino acid sequence (epitope-tag), and b) the aminoacid sequence of an α- and/or β-chain of a TCR as defined above, whereinsaid epitope-tag is selected from an epitope-tag added to the N- and/orC-terminus of said α- and/or β-chain or added into the α- and/or β-chainsequence, but outside the CDR3 region, an epitope-tag inserted into aconstant region of said α- and/or β-chain, and an epitope-tag replacinga number of amino acids in a constant region of said α- and/or β-chain;and a TCR comprising at least one fusion protein as above on the surfaceof the host cell; comprising contacting host cells in a sample with abinding agent that immunologically binds to the epitope-tag, andselection of said host cells based on said binding.

The present invention further provides an immunoglobulin molecule,anticaline, TCR γ/δ chain having a CDR3 region as defined herein (or aderivative thereof) inserted.

In a third aspect, the invention is directed to a T cell expressing aTCR as defined herein or a TCR comprising one of the CDR3 regions asdefined above.

Furthermore, the invention provides a vector, preferably a plasmid,shuttle vector, phagemide, cosmid, expression vector, retroviral vector,adenoviral vector or particle and/or vector to be used in gene therapy,which comprises one or more of the nucleic acids as disclosed above.

In the context of the present invention, a “vector” shall mean a nucleicacid molecule as introduced into a host cell, thereby producing atransformed host cell. A vector may include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector may also include one or more selectable marker genes and othergenetic elements known to those of ordinary skill in the art. A vectorpreferably is an expression vector that includes a nucleic acidaccording to the present invention operably linked to sequences allowingfor the expression of said nucleic acid.

A fourth aspect provides a cell, preferably a peripheral bloodlymphocyte (PBL) which has been transformed with the above vector. Thestep of cloning the T cell receptor (TCR) of the isolated T cells and/orexpressing the TCR transgenes in PBMC can be done according toestablished methods such as those described in Engels et al., 2005.

In a fifth aspect, the present invention provides a pharmaceuticalcomposition which comprises a TCR, a T cell or cell (PBL) as definedabove and a pharmaceutically acceptable carrier.

Those active components of the present invention are preferably used insuch a pharmaceutical composition, in doses mixed with an acceptablecarrier or carrier material, that the disease can be treated or at leastalleviated. Such a composition can (in addition to the active componentand the carrier) include filling material, salts, buffer, stabilizers,solubilizers and other materials, which are known state of the art.

The term “pharmaceutically acceptable” defines a non-toxic material,which does not interfere with effectiveness of the biological activityof the active component. The choice of the carrier is dependent on theapplication.

The pharmaceutical composition can contain additional components whichenhance the activity of the active component or which supplement thetreatment. Such additional components and/or factors can be part of thepharmaceutical composition to achieve synergistic effects or to minimizeadverse or unwanted effects.

Techniques for the formulation or preparation and application/medicationof active components of the present invention are published in“Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa.,latest edition. An appropriate application is a parenteral application,for example intramuscular, subcutaneous, intramedular injections as wellas intrathecal, direct intraventricular, intravenous, intranodal,intraperitoneal or intratumoral injections. The intravenous injection isthe preferred treatment of a patient.

According to a preferred embodiment, the pharmaceutical composition isan infusion or an injection.

An injectable composition is a pharmaceutically acceptable fluidcomposition comprising at least one active ingredient, e.g., an expandedT-cell population (for example autologous or allogenic to the patient tobe treated) expressing a TCR. The active ingredient is usually dissolvedor suspended in a physiologically acceptable carrier, and thecomposition can additionally comprise minor amounts of one or morenon-toxic auxiliary substances, such as emulsifying agents,preservatives, and pH buffering agents and the like. Such injectablecompositions that are useful for use with the fusion proteins of thisdisclosure are conventional; appropriate formulations are well known tothose of ordinary skill in the art.

In a further aspect, the present invention is directed to a method oftreating a patient in need of adoptive cell therapy, said methodcomprising administering to said patient a pharmaceutical composition asdefined above to said patient. The patient to be treated preferablybelongs to the group of HLA-A2 positive patients.

Preferably, said patient suffers from a disease involving malignantcells expressing tyrosinase, preferably melanoma, glioma, glioblastoma,and/or rare tumors of ectodermal origin.

The present invention now will be illustrated by the enclosed Figuresand the EXAMPLES. The following EXAMPLES further illustrate theinvention but, of course, should not be construed as limiting its scope.

DESCRIPTION OF THE FIGURES

FIG. 1: Screening of clones obtained from limiting dilution culturesafter DC priming. T cells were primed with dendritic cells expressingHLA-A2 and tyrosinase RNA. After two rounds of priming in vitro, cellswere cloned by limiting dilution. 14 to 28 days later T cell clonesshowing adequate growth in individual culture wells were identified bylight microscopy. Aliquots of growing clones were obtained and tested ina standard ⁵¹Cr release assay to measure their killing activity againsttwo melanoma target cell lines. Mel-A375 cells express HLA-A2 but nottyrosinase. Mel-93.04A12 cells express HLA-A2 and tyrosinase, so theycan form the ligands recognized by HLA-A2-restricted, tyrosinase peptide(YMDGTMSQV; SEQ ID NO: 9)-specific T cells. If Mel-A375 cells arerecognized by T cell clones, this means the clones are alloreactive andrecognize HLA-A2 independent of tyrosinase peptide (i.e., clone T41 andT42). If the T cell clones only recognize Mel-93.04A12, then they shouldhave specificity for HLA-A2-tyrosinase peptide ligands (i.e. T58, T43).Percentage specific lysis mediated by various T cell clones, (listed onx-axis) is given for the two target melanoma cell lines. The arrowdesignates clone T58 which shows strong killing of Mel-93.04A12 but notof Mel-A375. This clone was selected for further characterization basedon its strong growth capacity.

FIGS. 2A-2D: Comparison of clones T58 and IVS-B

FIG. 2A: Cytotoxic activity directed against melanoma cell lines.

The killing capacity of clone T58 was compared with that of clone IVS-B,derived from a melanoma patient, using as target cells the T2 cell linepulsed with synthetic tyrosinase− peptide for the amino acid sequenceYMDGTMSQV (SEQ ID NO: 9) in different molar concentrations, listed onthe x-axis. The % relative lysis is given on the y-axis. Theconcentration of peptide that corresponds to 50% relative lysis isindicated by the crossing lines and shows that clone T58 can recognizesubstantially lower concentrations of peptide in comparison to cloneIVS-B.

FIG. 2B: Measurement of multimer binding and off-rates.

The two clones were incubated with multimers to determine the percentageof positive cells at time 0 h. Both clones bound multimer on 100% of thecells. Multimer was washed out and the clones were incubated in mediumcontaining HLA-A2-specific antibody. When multimers are released fromthe cell surface, they are captured by the antibody and can not rebindto the cells. The percent multimer-positive cells were reanalyzed at 1 hand 2 h.

FIG. 2C: Interferon-gamma secretion after stimulation with melanoma celllines. Clone T58 and IVS-B were co-cultured with the two melanoma celllines used for the initial screening (described in FIG. 1) and theirsecretion of IFN-γ into the culture medium was assessed by standardELISA after 24 hours. n.d.=not detectable. Data are presented as pg/mlon the y-axis.

FIG. 2D: Cytotoxic activity against melanoma cell lines.

The clones were compared for killing activity using a standard⁵¹Cr-release assay as described in FIG. 1. Data are given as percentspecific lysis on the y-axis.

FIGS. 3A-3C: Recognition of primary melanoma tumor cells by clone T58and IVS-B.

(FIG. 3A) HLA-A2 surface expression on primary tumor cells (passage 12)of an HLA-A2⁻ melanoma patient transfected with 50 μg HLA-A2 ivt-RNA andon established melanoma cell lines Mel-93.04A12 (HLA-A2⁺trosinase⁺) andMel-A375 (HLA-A2⁺tyrosinase⁻) was measured by flow cytometry afterstaining with HLA-A2-specific monoclonal antibody. Each histogram showsthe stained sample (filled curves) and the corresponding control sample(empty curves): control curves represent untransfected primary tumorcells stained with HLA-A2-specific monoclonal antibody (left histogram)or melanoma cell lines stained with isotype control antibody. HLA-A2protein expression on RNA-transfected primary tumor cells was detected10 h after electroporation. (FIG. 3B) The capacity of thepatient-derived T cell clone (IVS-B), and T cell clone T58 to secreteIFN-γ or (FIG. 3C) release perforin in co-culture with the melanomacells shown above was measured in ELISPOT assays.

FIGS. 4A-4D: Transfer of antigen specificity by TCR retroviral genetransfer. (FIG. 4A) The human TCR-deficient T cell line Jurkat76⁹ wastransduced with the TCR of the T cell clone T58. TCR-expression wasdetected using tyrosinase-peptide-specific HLA-multimers. TCR expressionwas only detected in Jurkat76 cells tranduced with TCR-T58 (righthistogram) and not in untransduced Jurkat76 cells (left histogram).(FIG. 4B) PBL of a healthy donor were retrovirally transduced withTCR-T58. After 10 days, untransduced and TCR-transduced PBL wereanalysed for tyrosinase TCR-expression using specific HLA-multimers.Multimer staining is shown on the x-axis and CD8 staining on the y-axis.The percentage of multimer⁺CD8⁺ T cells is displayed in the upper rightquadrant. (FIG. 4C) Functionality of TCR-transduced PBL was measuredusing a standard IFN-γ release assay. T2 cells loaded with gradedamounts of tyrosinase₃₆₉₋₃₇₇ peptide (YMDGTMSQV; SEQ ID NO: 9; 10⁻¹²M-10⁻⁵ M) were used as target cells at a fixed effector to target cellratio of 1:1. Untransduced PBL served as a control and showed notyrosinase-peptide specific IFN-γ release (data not shown). Data areshown as pg/ml cytokine after subtration of secretion by untransducedPBL controls. (FIG. 4D) The capacity to secrete IFN-γ in co-culture withmelanoma cell lines SK-Mel-28 (HLA-A2⁻tyrosinase⁺), Mel-A375(HLA-A2⁺tyrosinase⁻), Mel-624.38 (HLA-A2⁺tyrosinase⁺) and Mel-93.04A12(HLA-A2⁺tyrosinase⁺) was assessed using a standard IFN-γ release assayusing an E:T=1:1; (n.d.=not detectable).

FIGS. 5A-5B: Transfer of specificity of T58 and IVS-B for HLA-A2 andtyrosinase− peptide YMDGTMSQV (SEQ ID NO: 9) by TCR retroviral genetransfer. (FIG. 5A) PBL of a healthy donor were retrovirally transducedwith the patient-derived TCR—IVS—B or the TCR-T58. After 11 days,untransduced and TCR-transduced PBL were analysed for tyrosinaseTCR-expression using specific HLA-multimers. Multimer staining is shownon the x-axis and CD8 staining on the y-axis. The percentage ofmultimer⁺CD8⁺ T cells is displayed in the upper right quadrant. (FIG.5B) Functionality of TCR-transduced PBL was measured using a standardIFN-γ release assay. T2 cells loaded with graded amounts oftyrosinase₃₆₉₋₃₇₇ peptide (10⁻¹¹ M-10⁻⁵ M) or with 10⁻⁵ M irrelevantinfluenza matrix protein₅₈₋₆₆ were used as target cells at a fixedeffector to target cell ratio of 1:1. Untransduced PBL served as acontrol and showed no tyrosinase-peptide specific IFN-γ release (datanot shown). Data are shown as pg/ml cytokine after substration ofsecretion by untransduced PBL controls (mean=318 pg/ml; range=219-368pg/ml) and adjustment for comparable numbers of multimer⁺ cells.

FIG. 6: Tyrosinase peptide-specific CTL recognition of tumor cell linesand primary melanoma tumor cells. Columns represent the amount of IFN-γ(pg/ml) secreted by self-restricted D115 CTL and allo-restricted T58 CTLin co-culture with a panel of tumor cell lines from left to right: MaCa1(HLA-A2-tyrosinase−); SK-Mel-28 (HLA-A2-tyrosinase+); Mel-A375, RCC-26,PancTu 1, MaCa1/A2, and UTS CC 1588 (all HLA-A2+tyrosinase−);Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29 and WM-266-4 (allHLA-A2+tyrosinase+). T cells designates CTL without stimulating cells.The HLA-A2+tyrosinase− tumor cell lines Mel-A375, RCC-26 and MaCa1/A2were exogenously loaded with either 10-5 M irrelevant flu peptide or10-5 M tyrosinase peptide YMD and IFN-γ secretion was measured by ELISAand given as pg/ml.

FIG. 7: Transfer of antigen specificity by retroviral transfer ofTCR-D115 and TCR-T58. PBL of a healthy donor were transduced withTCR-D115 or TCR-T58. Specificity of recognition was assessed by IFN-γrelease following co-culture with the tumor cell lines from left toright: MaCa1 (HLA-A2-tyrosinase−); SK-Mel-28 (HLA-A2-tyrosinase+);Mel-A375, RCC-26, PancTu 1, MaCa1/A2, and UTS CC 1588 (allHLA-A2+tyrosinase−); Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29 andWM-266-4 (all HLA-A2+tyrosinase+). T designates CTL without stimulatingcells. The HLA-A2+tyrosinase− tumor cell lines Mel-A375, RCC-26 andMaCa1/A2 were exogenously loaded with either 10-5 M irrelevant flupeptide or 10-5 M tyrosinase peptide YMD and IFN-γ secretion wasmeasured by ELISA and given as pg/ml.

FIGS. 8A-8F: Transfer of antigen specificity by retroviral transfer ofTCR-D115 and TCR-T58. (FIG. 8A) PBL of a healthy donor were transducedwith TCR-D115 or TCR-T58. Unsorted TCR-transduced PBL were analyzed onday 10 for transgenic TCR-expression using irrelevant B7-pp65 andA2-pp65 multimers and specific A2-tyr multimers. Untransduced PBL showedno multimer binding (0.1%, data not shown). Percentages of multimer+CD8+T cells are displayed in the upper right quadrant. (FIGS. 8A and 8B)show the IFN-γ release of unsorted TCR-transduced PBL followingstimulation with T2 cells loaded with graded amounts of tyrosinasepeptide (10-12 M-10-5 M) at a ratio of 2:1. In FIG. 8B the relativeIFN-γ release is displayed in percent and in FIG. 8C the specific IFN-γrelease is presented as pg/ml. (FIG. 8D) Functionality of unsortedTCR-transduced PBL was measured by IFN-γ release using autologousHLA-A2+PBMC loaded with tyrosinase peptide (10-11 M-10-6 M) asstimulating cells at ratio of 2:1. Untransduced PBL (▴) showed nopeptide-specific IFN-γ release. (FIG. 8E) The HLA-A2+tyrosinase− tumorcell lines Mel-A375, RCC-26 and MaCa1/A2 were exogenously loaded witheither 10-5 M irrelevant flu peptide (f) or 10-5 M tyrosinase peptideYMD (t) and IFN-γ secretion was measured by ELISA and given as pg/ml.(FIG. 8F) Specificity of recognition was assessed by IFN-γ releasefollowing co-culture with the tumor cell lines from left to right: MaCa1(HLA-A2-tyrosinase−); SK-Mel-28 (HLA-A2-tyrosinase+); Mel-A375, RCC-26,PancTu 1, MaCa1/A2, and UTS CC 1588 (all HLA-A2+tyrosinase−);Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29 and WM-266-4 (allHLA-A2+tyrosinase+). T designates CTL without stimulating cells. FIGS.9A-9D: TCR transfer retains differences in cytokine profile. On the lefthand side of FIGS. 9A-9D, the cytokine release of TCR-transduced PBL inco-culture with the melanoma lines Mel-A375 (HLA-A2+tyrosinase−) andMel-624.38 (HLA-A2+tyrosinase+) is depicted, on the right hand side thecorresponding cytokine release after stimulation with T2 cells loadedwith graded amounts of tyrosinase peptide (10-12 M-10-5 M) is shown.Untransduced PBL (▴) showed no peptide-specific cytokine release. Thefollowing cytokines were measured: IFN-γ (FIG. 9A), IL-2 (FIG. 9B),TNF-α (FIG. 9C) and MIP-1f3 (FIG. 9D). The levels of cytokine secretionfor all four cytokines were higher when PBL transduced with theallo-restricted TCR-T58 were used. Since untransduced PBL secreted veryhigh levels of MIP-1β in response to T2 cells the peptide titration forthis cytokine could not be evaluated.

EXAMPLE 1

The inventors prepared stimulating dendritic cells (DC) from anHLA-A2-negative healthy donor that co-expressed allogeneicHLA-A*0201-molecules and tyrosinase protein using mature DC that wereelectroporated with in vitro transcribed (ivt)-RNA for tyrosinase andHLA-A2, as described^(1,2). These DC were used to prime purified,autologous CD8⁺ T cells using two rounds of stimulation with freshlyprepared DC. After these two rounds of priming, CD8⁺ T cells with T cellreceptors (TCR) recognizing HLA-A2-tyrosinase₃₆₉₋₃₇₇-peptide complexeswere stained using a tyrosinase₃₆₉₋₃₇₇/HLA-A*0201-multimer³.CD8⁺multimer⁺ cells were isolated by fluorescence activated cellsorting. Sorted cells were cloned in limiting dilution cultures andisolated clones showing HLA-A2/tyrosinase-peptide specificity wereexpanded using antigen-independent stimulation⁴. The T cell clone T58was identified in an initial screen as having good functional activity(FIG. 1).

Because T58 was isolated from an HLA-A*0201-negative donor it representsan allo-restricted T cell clone that did not undergo negative selectionin vivo. The activity of the T58 clone was compared with the IVS-B clonethat was isolated from a patient with metastatic melanoma⁵. This clonerecognizes exactly the same HLA-A2/tyrosinase peptide ligand as cloneT58 but it is self-restricted since it was activated in vivo in thepatient who was HLA-A*0201-positive. This patient-derived T cell clonerepresents an example of T cells that are available in the peripheralrepertoire that have undergone negative selection againstself-peptides/self-MHC-molecules in the thymus in vivo.

Side-by-side comparisons of clone T58 and clone IVS-B were made todemonstrate the superior properties of the allo-restricted T58 cloneversus the self-restricted IVS-B clone. Functional T cell avidity fortyrosinase₃₆₉₋₃₇₇ peptide recognition was measured in a ⁵¹Cr-releaseassay using HLA-A2⁺ T2 cells pulsed with graded amounts of exogenouspeptide as target cells. The peptide concentration needed for 50%relative lysis defined the value of half-maximum lysis⁶. Theallo-restricted T cell clone T58 required substantially less peptide tobe activated by peptide-pulsed T2 cells than clone IVS-B (6.0×10⁻¹⁰ Mvs. 3.0×10⁻⁸ M) (FIG. 2A).

As an estimate of structural TCR-MHC/peptide binding affinity, loss ofmultimer binding was measured over time (i.e. HLA-multimer off-rate). Aslower off-rate indicates that TCR-ligand interactions are more stableand of higher structural affinity⁷. After initial incubation withmultimer and washing, T cells were incubated for 1 h and 2 h withoutmultimers in the presence of HLA-A2-specific antibody to preventcellular re-association of released multimers. The melanomapatient-derived T cell clone IVS-B showed an intermediate multimerbinding: all cells were multimer⁺ at 0 h and about 40% retainedmultimers at 1 and 2 h (FIG. 2B). In contrast, clone T58 had a sloweroff-rate, showing 74% positive binding at 1 h versus 41% for clone IVS-Band even at 2 h still had somewhat more multimer⁺ cells (55% vs. 40%).

Both T cell clones were analyzed in an IFN-γ release assay for functionand specificity (FIG. 2C). The clones were co-cultured with two melanomacell lines that express HLA-A2 molecules but differ with respect toexpression of tyrosinase protein: Mel-93.04A12 co-expresses bothproteins (HLA-A2⁺tyrosinase⁺) but Mel-A375 fails to express tyrosinaseprotein (HLA-A2⁺tyrosinase⁻) and therefore can not generate theMHC-peptide ligand seen by the T cell clones. Allo-restricted T cellclone T58 was induced to secrete a high level of IFN-γ by thetyrosinase-expressing melanoma cell line, whereas only marginal cytokinesecretion was seen with IVS-B cells (1,234 pg/ml vs. 106 pg/ml),demonstrating the vastly superior function of clone T58 in recognizingtumor cells expressing their HLA-A2-tyrosinase ligand. As expected, theclones showed no detectable IFN-γ secretion after stimulation withMel-A375 cells, demonstrating the specificity for HLA-A2 and tyrosinaseexpression for tumor cell recognition.

The killing capacity of allo-restricted clone T58 was also compared withclone IVS-B using a ⁵¹Cr-release assay (FIG. 2D). Again, clone T58showed superior function (76% vs. 24% specific lysis).

Both clones were also tested for their capacity to recognize primarymelanoma cells. Since primary HLA-A2+melanoma cells were not available,we introduced ivt-RNA for HLA-A2 into the tumor cells as for DC (FIG.3A). Function was measured using ELISPOT assays detecting IFN-γsecretion and perforin release to bypass high spontaneous release ofradioactive label by primary tumor cells. Recognition of primary tumorcells was shown to be HLA-A2-restricted since primary tumor cellslacking HLA-A2 RNA were not recognized. Again, a strong difference wasobserved with poor recognition by the patient self-restricted IVS-Bcells versus good recognition by allo-restricted T58 cells as assessedwith IFN-γ secretion (FIG. 3B) and by perform secretion (FIG. 3C).

To demonstrate that the superior functional avidity of allo-restrictedT58 cells resided directly in the TCR, separate recombinant retroviruseswere created for TCR alpha and beta chains of clone T58 as described⁸.Human TCR-deficient Jurkat76 cells⁹ were co-infected with the α-chainand β-chain retroviruses and transgenic TCR-expression was measured bymultimer staining. TCR-T58 was expressed at a good level, demonstratingadequate quality of the separate retroviral supernatants (FIG. 4A).Next, activated peripheral blood lymphocytes (PBL) of a healthyHLA-A2⁻donor were transduced and analyzed with multimers fortyrosinase-specific TCR-expression (FIG. 4B). Despite this lowfrequency, PBL transduced with TCR-T58 released high amounts of IFN-γfollowing stimulation with T2 cells pulsed with graded amounts oftyrosinase-peptide (FIG. 4C). TCR-T58 transduced PBL could also respondspecifically to stimulation by melanoma cell lines that expressed HLA-A2and tyrosinase (FIG. 4D). They did not respond to tumor cells that didnot express HLA-A2 or tyrosinase, again demonstrating the specificity ofHLA-A2-tyrosinase ligands for T58 recognition.

Bi-cistronic retroviral vectors were also prepared encoding the α-chainand β-chains of the TCR of IVS-B cells and used to transduce activatedPBL. In parallel, the same activated PBL were transduced withbi-cistronic retroviral vectors encoding the two chains of TCR-T58. PBLexpressing the corresponding receptors were identified by co-stainingfor CD8 and multimer and showed low numbers of positive cells. (FIG. 5A)Despite their low frequency, PBL transduced with TCR-T58 released highamounts of IFN-γ following stimulation with T2 cells pulsed with gradedamounts of tyrosinase− peptide. PBL expressing TCR—IVS-B secreted farless IFN-γ. Tyrosinase peptide-specific cytokine secretion was notdetected with untransduced PBL control cells. Data are shown as pg/mlcytokine after substraction of secretion by untransduced PBL controls(mean=318; range=219-369 pg/ml) (FIG. 5B).

Table 1 shows the genetic information regarding the use of VJ and VDJgene segments by the alpha and beta chains of TCR-T58, respectively. TheCDR3 regions, according to IMGT, are presented as nucleotide sequencesand amino acid sequences. Also shown are the codon optimized sequencesfor the full VJ and VDJ regions.

Materials and Methods Cell Lines

The human melanoma cell lines, Mel-A375 (HLA-A2⁺, tyrosinase⁻; CRL-1619,American Type Culture Collection (ATCC), Bethesda, Md.), Mel-93.04A12(HLA-A2+, tyrosinase⁺, gift of P. Schrier, Department ofImmunohematology, Leiden University Hospital, The Netherlands),Mel-624.38¹⁰ (HLA-A2⁺, tyrosinase⁺, gift of M. C. Panelli, NationalInstitutes of Health, Bethesda, Md.), SK-Mel-28 (HLA-A2⁻, tyrosinase⁺;MTB-72, ATCC) as well as the lymphoid cell line T2 (CRL-1992, ATCC), andthe human TCR-deficient Jurkat76⁹ T cell line were cultured in RPMI 1640medium supplemented with 12% fetal bovine serum (FBS), 2 mM L-glutamineand 1 mM sodium-pyruvate and non-essential amino acids.

The HLA-A*0201-restricted tyrosinase₃₆₉₋₃₇₇ peptide-specific melanomapatient-derived IVS-B T cell clone was cultured as described⁵.

Production of Tyrosinase and HLA-A2 Ivt-RNA

The plasmid pCDM8-HLA-A2 with HLA-A*0201 cDNA and the pZeoSV2+/huTyrwith tyrosinase cDNA were linearized and used as in vitro transcriptiontemplates to produce RNA with the aid of the mMESSAGE mMACHINE T7 kit(Ambion, Austin, Tex.) according to the manufacturer's instructions.

De Novo Priming of T Cells with RNA-Pulsed DC

Blood samples from healthy donors were collected after informed consentand with approval of the Institutional Review Board of the UniversityHospital of the Ludwig-Maximilians-University, Münich, Germany.Peripheral blood lymphocytes (PBL) were isolated by Ficoll densitygradient centrifugation. PBL were resuspended in 15 ml very lowendotoxin (VLE) RPMI 1640 medium (Biochrom, Berlin, Germany)supplemented with 1.5% human serum (DC medium) at 7.5×10⁷ cells per 75cm² culture flask and incubated at 37° C. and 5% CO2 for 1 h.Non-adherent cells were carefully removed by washing. Mature DC wereprepared from adherent monocytes and transfected with ivt-RNA viaelectroporation as previously described². DC of HLA-A2⁻donors wereco-transfected with 24 μg tyrosinase ivt-RNA and 48 μg HLA-A2 ivt-RNA.On the same day, autologous CD8⁺ T lymphocytes were enriched from PBLvia negative selection using a commercial kit according to themanufacturer's instructions (CD8⁺ T cell Isolation Kit II (human),Miltenyi, Bergisch Gladbach, Germany). Co-cultures were initiated 10 hafter DC electroporation in 24-well plates (TPP, Trasadingen,Switzerland) by adding 1×10⁵ RNA-pulsed DC to 1×10⁶ CD8⁺ T cells in RPMI1640, supplemented with 10% heat-inactivated human serum, 4 mML-glutamine, 12.5 mM HEPES, 50 (1M β-mercaptoethanol and 100 U/mlpenicillin/streptomycin (T cell medium). IL-7 (5 ng/ml) (Promokine,Heidelberg, Germany) was added on day 0 and 50 U/ml IL-2 (ChironBehring, Marburg, Germany) was added after 2 days and then on every3^(rd) subsequent day. Addition of IL-2 was delayed to decreaseproliferation of non-specific CD8⁺ T cells⁴. The 2^(nd) stimulation ofprimed T cells was made after seven days using freshly preparedRNA-pulsed DC.

HLA-Multimer Staining and Sorting

Seven days after the 2^(nd) stimulation of CD8-enriched T cells withRNA-pulsed DC, HLA-A2-restricted tyrosinase-specific T cells weredetected by staining with a PE-labeled HLA-A*0201/htyr₃₆₉₋₃₇₇peptide/human β₂m multimer¹¹, anti-CD8-APC antibody (clone RPA-T8, BDPharmingen, Franklin Lakes, N.J.) and propidium iodide (PI: 2 μg/ml).For sorting, up to 5×10⁶ cells were incubated with 12 μg multimer in 100μl PBS+0.5% human serum. CD8-APC antibody was then added at 1/50 for anadditional 25 min. After staining cells were washed twice and diluted inPBS+0.5% human serum with PI for sorting. 20-50×10⁶ total cells perpriming culture were stained for sorting. PI-negative cells were gatedand CD8⁺multimer⁺ T cells were sorted on a FACSAria cell sorter (BDBiosciences) with a 70 μm nozzle, at a rate of 15,000 events/s.

For HLA-multimer off-rate assays, cells were washed after multimerbinding and resuspended in FACS buffer containing saturating amounts ofBB7.2 monoclonal antibody (ATCC) to capture detached multimers andprevent rebinding to T cells. After 1 or 2 h, samples were fixed in FACSbuffer with 1% paraformaldehyde and analysed by flow cytometry.

Culture of Peptide-Specific T Clones

Multimer-sorted T cells were cloned by limiting dilution. Clones wereplated in 96-well round-bottom plates (TPP) in 200 μl/well T cellmedium. 50 IU/ml IL-2 was supplemented every 3 days with 5 ng/ml IL-7and 10 ng/ml IL-15 (PeproTech Inc., Rocky Hill, N.J.) every 7 days. Tcell clones were stimulated non-specifically with anti-CD3 antibody (0.1μg/ml; OKT-3) and provided with 1×10⁵ feeder cells per 96-well,consisting of irradiated (50 Gy) PBL derived from a pool of fiveunrelated donors and 1×10⁴ irradiated (150 Gy) EBV-transformedallogeneic B-LCL every two weeks. Proliferating T cells were transferredinto 24-well plates (TPP) and cultured in 1.5 ml T cell medium pluscytokines. 1×10⁶ allogeneic irradiated PBL and 1×10⁵ irradiatedEBV-transformed allogeneic B-LCL were added per well as feeder cells in24-well plates. Clonality was determined by TCR-beta-chain receptoranalysis, as described¹².

Peptide Loading of T2 Cells

For exogenous peptide pulsing, 1×10⁶ T2 cells were incubated at 37° C.and 5% CO2 for 2 h with 10 μg/ml human β2-microglobulin (Calbiochem, SanDiego, Calif.) and titrating amounts, ranging from 10⁻⁵ M to 10⁻¹² M, ofthe tyrosinase peptide YMD (tyrosinase₃₆₉₋₃₇₇ YMDGTMSQV, SEQ ID NO: 9,Metabion, Martinsried, Germany). T2 cells pulsed with 10⁻⁵ M influenzapeptide GIL (influenza matrix protein₅₈₋₆₆ GILGFVTL, SEQ ID NO: 10,Metabion) served as negative control. After washing, peptide-loaded T2cells were used as target cells in cytotoxicity or IFN-γ-release assays.

IFN-γ Release Assay

For investigation of specificity, T cell clones (2×10³ cells in 100 μl)were incubated with the respective melanoma cell lines or peptide-pulsedT2 cells (1×10⁴ cells in 100 μl). Culture supernatants were harvestedafter 24 h co-culture and assessed by a standard ELISA using the OptEIA™Human IFN-γ Set (BD Biosciences Pharmingen).

Cytotoxicity Assay

Cytotoxic activity of T cell clones was analysed in a standard 4 h51-chromium release assay. Melanoma cells or peptide-loaded T2 cellswere used as target cells. Briefly, 1×10⁶ target cells were labeled with100 μCi Na₂ ⁵¹CrO4 (ICN Biochemicals, Irvine, Calif.) for 1-1.5 h.⁵¹Cr-labeled target cells were cultured with T cells in 100 μl/well RPMI1640 with 12% FCS in V-bottom 96-well tissue culture plates (Greiner,Solingen, Germany). For determination of functional avidity 1×10⁴ Tcells were added to 1×10³ peptide-pulsed T2 cells loaded with titratedamounts of peptide, giving a constant E:T of 10:1.

After 4 h co-culture at 37° C., 50 μl of supernatant were collected andradioactivity was measured in a gamma counter. The percentage ofspecific lysis was calculated as: 100×(experimental release−spontaneousrelease)/(maximum release−spontaneous release). Spontaneous release wasassessed by incubating target cells in the absence of effector cells andwas generally less than 15%. For the calculation of percent relativelysis, the maximum percent specific lysis was set to the reference valueof 100% and corresponding values were calculated corresponding to thisreference. To determine half-maximum lysis, percent relative lysis wasplotted against peptide concentration. The peptide concentration atwhich the curve crossed 50% relative lysis was taken as the value ofhalf-maximum lysis⁶.

ELISPOT

Antibody pre-coated PVDF plates (Mabtech AB, Nacka, Sweden) wereincubated at 37° C. in CTL Test™ medium (Cellular Technology Ltd.,Cleveland, Ohio) for 2 h to block unspecific binding. For the IFN-γELISPOT, plates were pre-coated with the IFN-γ-specific capture antibodyclone 1-D1K; for perforin ELISPOT plates were pre-coated with theperforin-specific capture antibody (clone Pf-80/164; Mabtech AB). PrimedT cells were washed with CTL Wash™ Supplement culture medium (CellularTechnology Ltd) and 1×10³ responder T cells were stimulated with 5×10³melanoma cells in 150 μl CTL Test™ medium and 24 h later assessed inIFN-γ ELISPOT or 48 h later in perforin ELISPOT. After washing withPBS/0.01% Tween and PBS alone, plates were incubated either with adirect streptavidin-alkaline phosphatase (ALP)-conjugated detectionantibody (clone 7-B6-1; Mabtech AB) for IFN-γ ELISPOT or withbiotinylated detection antibody (clone Pf-344; Mabtech AB) for perforinELISPOT for 2 h at room temperature following a 1 h incubation withstreptavidin-alkaline phosphatase (ALP). The plates were washed againand a ready-to-use BCIP/NBT-plus substrate solution (Mabtech AB) wasadded. Spots were counted using the AID reader system ELRO3 with thesoftware version 4.0 (AID Autoimmun Diagnostika GmbH, Strassberg,Germany).

Construction of Retroviral Vectors, Production of Virus Supernatants andTransduction of Jurkat76 T Cells and PBL

For TCR identification of tumor-specific T cell clones, part of theTCRα- and TCRβ-chain sequences including the complementary determiningregion (CDR3) was amplified by PCR using a panel of TCRVα and TCRVβprimers combined with the respective constant region primer asdescribed¹³. The TCRα and TCRβ chain genes of T cell clones T58 andIVS-B were amplified by PCR with gene specific primers and cloned intothe retroviral vector MP71PRE⁸ via NotI and EcoRI restriction sites.Both chains of human TCR-T58 (Vα7, Vβ23) and TCR—IVS-B (Vα3, Vβ14) wereconstructed as single-TCR gene vectors or double-TCR gene vectors(pMP71-T58α and pMP71-T58β, pMP71-IVS-Ba and pMP71-IVS-Bβ;pMP71-T58β-P2A-T58α and pMP71-IVS-Bβ-P2A-IVS-Bα). Retroviral vectorplasmids were co-transfected into 293T cells with expression plasmidsencoding Moloney MLV gag/pol and MLV-10A1 env gene to produceamphotropic MLV-pseudotyped retroviruses as described¹⁴. The humanTCR-deficient T cell line Jurkat76 and PBL were transduced asreported¹⁴. Jurkat76 cells (5 days after transduction) and PBL (10 daysafter transduction) were stained using PE-labeled HLA-A*0201/htyr₃₆₉₋₃₇₇peptide/human β₂m multimer and anti-CD8-FITC antibody. On day 13 anIFN-γ release assay was performed using T2 cells loaded with gradedamounts of tyrosinase₃₆₉₋₃₇₇ peptide (10⁻¹² M-10⁻⁵ M) or T2 cells pulsedwith 10⁻⁵ M influenza matrix protein₅₈₋₆₆ peptide and the tumor celllines SK-Mel-28, Mel-A375, Mel-624.38 and Mel-93.04A12 as stimulatingcells at an E:T ratio=1:1. Control values for peptide-stimulateduntransduced PBL were subtracted from values of transduced cells at eachpeptide concentration and then adjusted to comparable numbers of totalTCR-transgenic cells.

T58-TCR Analysis

For the T-cell receptor analysis of the tyrosinase-specific clone T58,part of the TCR alpha-chain and beta-chain containing the CDR3 regionwas amplified by RT-PCR using a panel of TCR Vα and TCR Vβ primerscombined with a respective TCR constant region primer. Products weresequenced and assigned according to IMGT (Table 1; IMGT, THEINTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®).

Modifications of the TCR-Sequence

Codon optimization of the VJ/VDJ-regions of both T58-TCR chains was doneto facilitate TCR mRNA translation (Table 1). Antibody-tags, for examplemyc-tags¹⁵ (Patent Application number: 06014606.5-1212) or othermodifications, for example a CD20 epitope, can be introduced in anyposition, i.e. the N-terminus of the TCRα-chain, that is recognized bythe depleting antibody and does not interfere with TCR-functionality.

TABLE 1TCR-CDR3 sequences and codon optimized VJ/VDJ regions of clone T58Alpha-chain VJ region* TRAV1-2 AJ28 CDR3 region* Nucleotide sequenceTGTGCTGTGACATACTCTGGGGCTGGGAGTTACCAAC TC (SEQ ID NO: 3)Amino acid sequence C A V T Y S G A G S Y Q L (SEQ ID NO: 5)Codon optimized VJ ATGTGGGGCGTGTTTCTGCTGTACGTGTCCATGAAGATGGGCGGCACCACCGGCCAGAACATCGACCAGCCCACCGAGATGACAGCCACCGAGGGCGCCATCGTGCAGATCAACTGCACCTACCAGACCAGCGGCTTCAACGGCCTGTTCTGGTATCAGCAGCACGCCGGCGAGGCCCCTACCTTCCTGAGCTACAACGTGCTGGACGGCCTGGAAGAGAAGGGCCGGTTCAGCAGCTTCCTGAGCCGGTCCAAGGGCTACAGCTACCTGCTGCTGAAAGAACTGCAGATGAAGGACAGCGCCAGCTACCTGTGCGCCGTGACCTACAGCGGAGCCGGCAGCTACCAGCTGACCTTCGGCAAG GGCACCAAGCTGTCCGTG (SEQ ID NO: 7)Beta-chain VDJ region* TRBV13 BD1 BJ1-4 CDR3 region* Nucleotide sequenceTGTGCCAGCAGTCAGAAACAGGGCTGGGAAAAAC TG (SEQ ID NO: 4) Amino acid sequenceC A S S Q K Q G W E K L (SEQ ID NO: 6) Codon optimized VDJATGCTGTCCCCCGATCTGCCCGACAGCGCCTGGAA CACCAGACTGCTGTGCCACGTGATGCTGTGTCTGCTGGGAGCCGGATCTGTGGCCGCTGGCGTGATCCA GAGCCCCAGACACCTGATCAAAGAGAAGCGGGAGACAGCCACCCTGAAGTGCTACCCCATCCCCCGGC ACGACACCGTGTACTGGTATCAGCAGGGACCAGGACAGGACCCCCAGTTCCTGATCAGCTTCTACGAGA AGATGCAGAGCGACAAGGGCAGCATCCCCGACAGATTCAGCGCCCAGCAGTTCAGCGACTACCACAGC GAGCTGAACATGAGCAGCCTGGAACTGGGCGACTCTGCCCTGTACTTCTGCGCCAGCAGCCAGAAGCA GGGCTGGGAGAAGCTGTTCTTCGGCAGCGGCACCCAGCTGTCCGTGCTG (SEQ ID NO: 8)

TCR alpha-chain (VJ region), TCR beta-chain (VDJ region) and CDR3lenghts are designated according to IMGT (IMGT, THE INTERNATIONALIMMUNOGENETICS INFORMATION SYSTEM®)

EXAMPLE 2

In EXAMPLE 1, data are provided that compared two T cell clones thatspecifically recognize a peptide derived from tyrosinase (i.e.,YMDGTMSQV (SEQ ID NO:9) hereafter referred to as YMD) presented byHLA-A*0201 molecules. The T cell clone T58 was an allo-restricted,peptide-specific T cell clone derived from an HLA-A2-negative donor. TheT cell clone IVS-B was derived from an HLA-A*0201-positive patient whosuffered from metastatic melanoma. This melanoma expressed tyrosinase.

In this EXAMPLE, comparisons have been extended to include an example ofa T cell clone, D115, which is also derived from an HLA-A*0201-positiveindividual and recognizes the same YMD peptide. However, in contrast toclone IVS-B, clone D115 was generated in vitro using responding T cellsderived from the blood of a healthy individual. Therefore, there havebeen no potential negative impacts on this T cell clone from a tumorenvironment (i.e., melanoma) in vivo.

FIG. 6 shows a comparison of the pattern of the target cell recognitionof the new clone D115 and clone T58 which is the subject of this patent.As can be clearly seen, both D115 and T58 show the same pattern ofrecognition, detected by secretion of interferon-gamma (y-axis), afterco-cultivation with various tumor cell lines (x-axis and figure legend).Neither clone recognizes tumor cells that are HLA-A2-negative butexpress tyrosinase, nor do they recognize tumor cells that areHLA-A2-positive and tyrosinase negative. On the other hand, both T cellclones recognize several tumor cell lines that are both HLA-A2-positiveand tyrosinase-positive. The role of the YMD peptide in this recognitionis shown by the finding that HLA-A2-positive tumor cells that do notexpress tyrosinase from which the YMD peptide could be processedinternally and transported to the cell surface by HLA-A2 molecules forpresentation, can be loaded with synthetic YMD peptide, leading to theirrecognition by D115 and T58. Thereby, both clones show the samespecificity for the YMD peptide presented by HLA-A2 molecules. However,the efficiency of recognition displayed by clone T58 is far superior toclone D115, as seen by the levels of interferon-gamma secretion. This,for example, leads to negligible recognition of the melanoma cell lineSK-Mel-29 by D115 but clear recognition by T58.

The TCR of clone D115 and T58 were expressed as recombinant proteins inactivated recipient lymphocytes (FIG. 7). When these TCR-transducedlymphocytes were retested with the same panel of target cells, theyshowed the same specificity pattern as the original T cell clones,demonstrating that the TCR recognition was responsible for the resultsseen in FIG. 6. Again, in FIG. 7 it is demonstrated that the TCR ofclone T58 shows superior recognition of the melanoma tumor cell linesthat express HLA-A2 and tyrosinase and the YMD peptide-pulsedHLA-A2-positive tumor cells.

FIG. 8A shows that the TCR-transduced lymphocytes show comparable levelsof expression of the respective recombinant TCRs, with each transducedpopulation having around 11% of T cells that bind a MHC multimercomprised of HLA-A2 molecules presenting the YMD peptide. Such bindingis not observed with control multimers that present other peptidesderived from the pp65 protein of human cytomegalovirus.

When the two populations of TCR-transduced PBL are stimulated withHLA-A2-positive antigen-presenting cells (i.e., T2 cells) that arepulsed with different concentrations of YMD peptide (shown on thex-axis), it can be seen that the cells expressing TCR-T58 release 50% oftheir maximal levels of interferon-gamma (y-axis) at 100-fold lowerpeptide concentrations. This peptide-sensitivity assay shows that theTCR-T58 has a much higher functional avidity when compared to TCR-D115(FIG. 8B).

This difference is further exemplified by the strong difference in themaximum levels of interferon-gamma produced by the TCR-T58- versusTCR-D115-transduced lymphocytes. In the case of TCR-T58 cells, themaximum reaches 5000 pg/ml whereas this results in only around 2000pg/ml for TCR-D115 in 24 hours. Furthermore, the amount of peptide thatmust be presented by T2 cells to cause release of 2000 pg/mlinterferon-gamma is 15,000-fold lower for triggering of this level ofresponse from TCR-T58-transduced lymphocytes compared withTCR-D115-transduced lymphocytes (FIG. 8C).

FIG. 8D shows another peptide-sensitivity assay, this time usingperipheral blood mononuclear cells that have been pulsed with titratingamounts of YMD peptide (x-axis). Once again, the amounts ofinterferon-gamma released by lymphocytes expressing TCR-T58 are muchgreater compared with TCR-D115. The arrows show that the first detectionof cytokine secretion occurs with 1000-fold less peptide for TCR-T58compared with TCR-D115.

FIGS. 8E and 8F demonstrate the specificity of the transduced lymphocytepopulations for peptide-pulsed tumor cells (FIG. 8E) or tumor cell linesexpressing HLA-A2 and tyrosinase (FIG. 8F). In all cases, recognition issuperior by lymphocytes expressing TCR-T58 compared to TCR-D115.

The superior secretion of cytokine is not limited to interferon-gamma.The levels of secretion of interleukin-2, TNF-alpha and MIP-lbeta arealso superior for TCR-T58. This is seen after stimulation of theTCR-transduced lymphocytes by tumor cells or by peptide-pulsed T2 cells(FIGS. 9A-9D).

Material and Methods Cell Lines

The human melanoma cell lines, Mel-A375 (HLA-A2⁺, tyrosinase⁻; CRL-1619,American Type Culture Collection (ATCC)), Mel-93.04A12 (HLA-A2⁺,tyrosinase⁺; gift of P. Schrier, Department of Immunohematology, LeidenUniversity Hospital, The Netherlands), Mel-624.38¹ and SK-Mel-23(HLA-A2⁺, tyrosinase⁺; gift of M. C. Panelli, National Institutes ofHealth, Bethesda, Md.), SK-Mel-28 (HLA-A2⁻, tyrosinase⁺; MTB-72, ATCC),SK-Mel-29 (HLA-A2⁺, tyrosinase⁺, gift of P. Rieber, Institute ofImmunology, Technical University Dresden, Germany), WM-266-4 (HLA-A2⁺,tyrosinase⁺; CRL-1676, ATCC) and primary cultures of a human melanoma(passage 6-12) and MaCa1 (HLA-A2⁻, tyrosinase⁻, gift of R. Wank, M. D.Münich, Germany), stable HLA-A*0201 transfectant of MaCa1 (MaCa1/A2)(HLA-A2+, tyrosinase⁻, gift of E. Noessner, Institute of MolecularImmunology, Helmholtz Zentrum Munchen, Germany), RCC-26² (HLA-A2⁺,tyrosinase⁻), PancTu1 (HLA-A2⁺, tyrosinase⁻, gift of P. Nelson,Department for Biological Chemistry University Hospital LMU Münich,Germany), UTS CC 1588 (HLA-A2⁺, tyrosinase⁻, gift of M. Schmitz,Institute of Immunology, Technical University Dresden, Germany) as wellas the lymphoid cell line T2 (CRL-1992, ATCC) were cultured in RPMI 1640medium supplemented with 12% fetal bovine serum (FBS), 2 mM L-glutamineand 1 mM sodium-pyruvate and non-essential amino acids.

Peptide Loading of T2 Cells, PBMC and Tumor Cells

For exogenous peptide pulsing, 1×10⁶ T2 cells were incubated at 37° C.and 5% CO2 for 2 h with 10 μg/ml human β₂-microglobulin (Calbiochem) andtitrating amounts, ranging from 10⁻⁵ M to 10⁻¹¹ M, of the tyrosinasepeptide YMD (tyrosinase₃₆₉₋₃₇₇ YMDGTMSQV, SEQ ID NO: 9, Metabion). T2cells pulsed with 10⁻⁵ M influenza peptide GIL (flu: influenza matrixprotein₅₈₋₆₆ GILGFVFTL, SEQ ID NO: 10, Metabion) served as the negativecontrol. PBMC were loaded with tyrosinase peptide as for T2 cells withtitrating amounts ranging from 10⁻⁶ to 10⁻¹¹ M. Tumor cells were loadedwith either 10⁻⁵ M flu peptide or 10⁻⁵ M tyrosinase peptide YMD asdescribed for T2 cells. After washing, peptide-loaded T2 cells, PBMC ortumor cells were used as stimulating cells in IFN-γ release assays.

Cytokine Assays

For investigation of specificity, CTL (2×10³ cells in 100 μl) wereincubated with various tumor cell lines (1×10⁴ cells in 100 μl), with orwithout peptide pulsing, as described above. Culture supernatants wereharvested after 24 h co-culture and assessed by a standard ELISA usingthe OptEIA™ Human IFN-γ Set (BD Biosciences). Data represent mean valueswith corresponding mean deviations calculated from duplicatedeterminations. For the calculation of % relative IFN-γ release, themaximum IFN-γ release was set to the reference value of 100% andcorresponding values were calculated corresponding to this reference.

To investigate multiple cytokines simultaneously (IFN-γ, IL-2, TNF-α andMIP-1β) cytokine secretion in supernatants of co-culture of CTL withtumor cells and with or without tyrosinase peptide pulsed T2 cells (10⁻⁵M) was measured using the multiplex protein array system technology(Bio-Rad Laboratories, Hercules, Calif.).

Retroviral TCR Gene Transfer

For TCR identification of tumor-specific CTL, regions of the TCRα- andTCRβ-chains encoding CDR3 were amplified by PCR using a panel of TCRVαand TCRVβ primers in combination with respective constant region primersas described.³ The full TCRα- and TCRβ-chain genes of CTL clones T58 andD115 were amplified by PCR using cDNA as template. Primer sequences willbe provided on request. The constant regions of both TCR chains wereexchanged by the murine counterparts to increase the stability of theTCR.⁴ The TCR chains were linked by a 2A peptide linker(TCRβ-P2A-TCRα)⁵, codon-optimized (Geneart)⁶ and cloned into theretroviral vector MP71PRE via NotI and EcoRI restriction sites.⁵Retroviral vector plasmids were co-transfected into 293T cells withexpression plasmids encoding Moloney MLV gag/pol and MLV-10A1 env gene,respectively, to produce amphotropic MLV-pseudotyped retroviruses asdescribed.⁵ Ten days after the second transduction, PBL were stainedusing PE-labeled A2-tyr multimer and FITC-labeled CD8-specific antibody.Multimers presenting peptides derived from cytomegalovirus pp65 wereused as controls: PE-labeled HLA-B7 pp65417-427 (B7-pp65) multimersserved as the HLA control and HLA-A2 pp65₄₉₅₋₅₀₃ multimers as apeptide-specificity control. On day 15 an IFN-γ release assay wasperformed using T2 cells or autologous PBMC loaded with graded amountsof tyrosinase peptide (10⁻¹² M-10⁻⁵ M) and the tumor cell lines MaCa1,SK-Mel-28, Mel-A375, RCC-26, PancTu 1, MaCa1/A2, UTS CC 1588,Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29 and WM-266-4 asstimulating cells at an E:T of 2:1.

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1-16. (canceled)
 17. A vector comprising a nucleic acid encoding a Tcell receptor (TCR) that binds to a tyrosinase peptide comprising theamino acid sequence of YMDGTMSQV (SEQ ID NO: 9), the nucleic acidcomprising the nucleic acid sequence of SEQ ID NO: 1 coding for theα-chain and/or the nucleic acid sequence of SEQ ID NO: 2 coding for theβ-chain of said TCR, or a derivative thereof, coding for the α- orβ-chain, wherein the chain has been altered by one or more additions,deletions, or conservative substitutions of from 1-15 amino acids, theadditions, deletions, or conservative substitutions being outside theCDR3 region of each chain, or a fragment thereof coding for a CDR3region of the TCR and having the nucleic acid sequence of SEQ ID NO: 3or 4 or coding for the amino acid sequences of SEQ ID NO: 5 or
 6. 18.The vector of claim 17, comprising the nucleic acid sequence of SEQ IDNO: 1 coding for the α-chain and/or the nucleic acid sequence of SEQ IDNO: 2 coding for the β-chain of said TCR.
 19. The vector of claim 18,comprising the nucleic acid sequence of SEQ ID NO: 1 coding for theα-chain and the nucleic acid sequence of SEQ ID NO: 2 coding for theβ-chain of said TCR.
 20. The vector of claim 17, wherein the derivativeof the α- or β-chain coding sequence is derived from SEQ ID NO: 1 and 2by codon optimization.
 21. The vector of claim 20, wherein thederivative of the α- or β-chain coding sequence comprises the nucleicacid sequence of SEQ ID NO: 7 coding for the α-chain and/or comprisesthe nucleic acid sequence of SEQ ID NO: 8 coding for the β-chain of saidTCR.
 22. The vector of claim 17, which is a plasmid, shuttle vector,phagemid, cosmid, expression vector, retroviral vector, adenoviralvector or particle and/or gene therapy vector.
 23. The vector of claim22, which is a plasmid.
 24. The vector of claim 22, which is aretroviral vector.
 25. The vector of claim 17, further comprising one ormore of (i) an origin of replication and (ii) one or more selectablemarker genes.
 26. A plasmid comprising a nucleic acid comprising thenucleic acid sequence of SEQ ID NO:
 2. 27. The plasmid of claim 26,further comprising an origin of replication and one or more selectablemarker genes.
 28. A vector comprising a nucleic acid comprising thenucleic acid sequences of SEQ ID NO: 3 and/or SEQ ID NO:4.
 29. A vectorcomprising a nucleic acid coding for a TCR binds to a tyrosinase peptidecomprising the amino acid sequence of YMDGTMSQV (SEQ ID NO: 9), the TCRcomprising the amino acid sequences of SEQ ID NO: 5 and/or 6.